Product Description

ISOLATE II PCR and Gel Kit is the simplest option for the purification of PCR products and for the isolation of DNA fragments from TAE and TBE agarose gel slices. A fast and easy-to-follow protocol is given for each application.

DNA fragments between 50 bp and 20 kb can be extracted from agarose gel slices in 20 minutes using a color indicator to help maintain optimal pH and identify undissolved agarose.

ISOLATE II PCR and Gel Kit has been designed to deliver optimal performance in downstream applications, including transformations, cloning, sequencing and restriction analysis.

Applications

Cloning

Ligation

Restriction digestion

Fluorescence sequencing

Labeling

PCR

Transfection

In vitro transcription

PCR product purification

5 kb, 1.2 kb and 500 bp PCR fragments were run on an agarose gel (lanes 1, 3 and 5) alongside the same products purified with ISOLATE II PCR and Gel Kit (lanes 2, 4 and 6). The results illustrate the ability of the ISOLATE II PCR and Gel Kit to remove small contaminants such as primers, primer-dimers, enzymes etc. without loss of the PCR product.

Recovery of DNA from agarose gel

5 kb, 1.2 kb, 500bp and 100 bp PCR fragments were run on an agarose gel (lanes O) alongside the same products run on 1% TAE agarose gel and extracted from the gel using ISOLATE II PCR and Gel Kit (lanes P). The results illustrate very high recovery rates of DNA fragments from agarose gels.

Shipping conditions

FAQs

The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.

Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.

Yes, though this may require some optimization. Warming the elution buffer to 50°C or even 70°C before applying it to the column can help to elute long PCR products, but there is the possibility using this adaptation to the protocol that the primers will be eluted too.

The ISOLATE II PCR and Gel Kit can be used under standard conditions to separate most PCR primers away from their product. A primer of only 30 bp is efficiently separated, but longer mutagenic primers of around 100 bp may be separated from the PCR buffer along with the amplicon.

The ISOLATE II Plasmid Mini Kit can be used to both purify and concentrate naked plasmid DNA. The DNA can be loaded directly onto a fresh column then the remaining washing, drying and elution steps followed as for the standard protocol. The sample can be concentrated by reducing the elution volume in the final stage. However, a better recovery can be obtained by using the ISOLATE II PCR and Gel Kit and treating the plasmid sample as though it were a PCR product.

For the optimal recovery of large RNA/DNA fragments it is necessary to optimize the elution step. To increase the recovery rate it would be helpful to use a larger volume for elution, incubate the column with the elution buffer prior to centrifugation and use repeated elution steps. To avoid excessive dilution it may be helpful to reapply the eluted fraction again. Furthermore it could be helpful for DNA elution to heat the elution buffer to 70°C.